Natural hirudin is a mixture of peptides composed of 65 or 66 amino acids and is secreted from salivary glands of medicinal leeches in very small amounts. A variant called HV-1 is the first hirudin isolated from the leeches. A variant called HV-2 differs from the aforesaid HV-1 in 9 amino acids, and HV-3 is identical with HV-2 up to the 32nd serine and differs in 10 amino acids including an additional 63rd alanine on the C-terminal end.
Further, the existence of tyrosine in which the phenolic hydroxide residue is sulfated as shown in the following formula ##STR1## has been confirmed.
It has been reported that the anti-thrombin activity increases about 10 times by the existence of sulfate on the tyrosine residue.
So far, it is very difficult to manufacture polypeptides having sulfated tyrosine in the molecule. When chemical introduction of a sulfate group on the tyrosine residue in polypeptides obtained by methods such as recombinant DNA technology is considered, in which case the amino acid sequence is long, it is very difficult to sulfate targeted tyrosine selectively without influencing other amino acids. It also requires drastic reaction conditions which often cause disruption of peptide bonds and it is difficult to obtain the sulfated compound in satisfactory yield. For this reason, hirudins under development at present as anti-coagulants are non-sulfated ones and have low activities.
Although the therapeutic application of hirudin as an anti-coagulant is thought to be effective, being a foreign compound, allergic symptoms such as shock and eczema are possible. However, it is thought to be possible to reduce allergic responses by decreasing the administration dose or shortening the amino acid sequence of the polypeptide. The aim of the present invention is to provide hirudin variants with higher anti-thrombin activity by sulfating the hydroxyl group of tyrosine residue in the molecule.